Summary ? Project 3: Deficiency of T-cell number and disorder of function result from hematopoietic stem cell therapy (HSCT), chemotherapy, and aging. The timely regeneration of T-cells and the simultaneous balanced reconstitution of the nave helper and effector T-cell subsets, along with the restoration of the T-cell repertoire remains a significant unmet clinical need in a number of other clinical settings. The goal of this project is to use bioengineered materials to augment or restore T cell immune function through de novo generation of T cells. It is based on prior work defining critical elements of the bone marrow microenvironment that enable T cell competence in hematopoietic stem and progenitor cells (HSPC), and decades of research developing strategies to engineer bony tissues. We hypothesize that biomaterials which promote bone formation and recreate key features of the normal HSC niche will enable more rapid reconstitution of T cell progenitors following HSCT. Engineering the expression of a particular Notch ligand, Dll-4, in this microenvironment is anticipated to greatly increase generation of a key T cell progenitor, the common lymphoid progenitor (CLP), and subsequently enhance reconstitution of T cell-mediated adaptive immunity. This hypothesis will be addressed with the following aims: (1) Develop cryogel-based biomimetics of the bone marrow microenvironment presenting DLL-4 to generate, in a minimally invasively manner, a HSPC niche to which transplanted HSPCs home, and enhance T cell neogenesis, (2) Define if a subset of HSPC home to the BMC, and if the BMC imposes selection on the HSPC with which it engages, and (3) Test the impact of the BMC on tumor antigen response in three host groups: following HSCT, chemotherapy and advanced age. These studies will determine if BMC introduction into murine hosts leads to increased generation of T cell progenitors (mouse and human), T competent cells entering the thymus, moving through thymocyte differentiation and providing improved T cell immunity. If successful, this project will provide a novel, clinically applicable means of augmenting anti-cancer immunity by improving T cell generation.